Involvement of the annexin A1-Fpr anti-inflammatory system in the ocular allergy.

Annexin A1 (ANXA1)-formyl peptide receptor (Fpr) system is potent effective mediators in the control of the inflammatory response. In this study, we evaluate the potential involvement of the Fpr family in the protective effect of the mimetic peptide of ANXA1 (ANXA12-26) using an experimental allergic conjunctivitis (AC) model in mice. Ovalbumin (OVA)/Alum-immunized wild-type (WT) and ANXA1-null (ANXA1-/-) Balb/c mice (days 0 and 7) were challenged by eye drops containing OVA on days 14-16, and two groups received ANXA12-26 alone or with Fpr antagonist Boc2 intraperitoneally during challenged days. As expected, plasma IgE anti-OVA levels increased significantly in the OVA-immunized WT and ANXA1-/- mice, supporting the efficacy of AC model. AC increased Fpr1 and Fpr2 levels in the conjunctiva and the lack of endogenous ANXA1 exacerbated Fpr2 expression only. In contrast, administering ANXA12-26 in the WT mice diminished Fpr2 levels in the conjunctiva, and the effect was reverted by Boc2. Ultrastructural analysis showed the co-localization of Fpr2 and ANXA1 in the plasma membrane of mast cells (MCs), eosinophils and neutrophils, supporting this system as being operative in the AC. Boc2 abrogated the ANXA12-26 effect by increasing the MC degranulation and the eosinophil influx in the conjunctiva, and these findings were supported by peroxidase eosinophil, eotaxin and MC protease levels. Additionally, the ANXA12-26-Fpr system in the AC was associated with the activation of ERK and JNK. Collectively, the data provided in vivo supports the anti-allergic effects of the ANXA1-Fpr system and may serve as a therapeutic target in this ocular disorder.

Galectin-3: role in ocular allergy and potential as a predictive biomarker.

AIMS: To evaluate galectin-3 (Gal-3), a ß-galactoside binding protein, as a possible biomarker in ocular allergy and further investigated the role of endogenous Gal-3 in a murine model of ovalbumin (OVA)-induced allergic conjunctivitis (AC). METHODS: Conjunctival impression cytology specimens from control and patients with severe vernal keratoconjunctivitis, treated or untreated, were used to evaluate Gal-3 expression by immunocytochemistry. To investigate the mechanism of action of Gal-3, OVA-immunised BALB/c male wild-type (WT) and Gal-3 null (Gal-3-/-) mice were challenged with eye drops containing OVA on days 14-16 with a subset of animals pretreated with 0.03% tacrolimus (TC) or dexamethasone (Dex). RESULTS: Patients with AC and OVA-sensitised WT mice exhibited increased levels of Gal-3 in the conjunctiva compared with control, an effect reverted by the action of Dex and TC therapy. Twenty-four hours after the final OVA challenge, total and anti-OVA IgE levels increased significantly in the blood of OVA-sensitised WT and Gal-3-/- mice compared with controls, supporting the efficacy of the AC model. The lack of endogenous Gal-3 exacerbated the local inflammatory response, increasing the influx of eosinophils and mast cell activation. Additionally, OVA-sensitised Gal-3-/- animals exhibited increased CD4+ expression in the eyes as well as eotaxin, IL-4, IL-13 and interferon-γ levels in the tear fluid compared with WT animals. CONCLUSION: Gal-3 contributes to the pathogenesis of ocular allergy and represents a relevant therapeutic target.

Evaluation of galectin-1 and galectin-3 as prospective biomarkers in keratoconus.

AIMS: To evaluate the expression of ß-galactoside-binding proteins galectin (Gal)-1 and Gal-3 in patients with keratoconus (KC) and postcorneal collagen cross-linking (CXL) treatment in vitro. METHODS: Tear fluid, cornea samples and conjunctival impression cytology specimens from control and KC patients were used to evaluate Gal-1 and Gal-3 expressions. Primary keratocytes were isolated by collagenase digestion from surgically removed corneas of five normal or KC human corneal buttons and cultured in Dulbecco's modified eagle medium/Ham's F12 medium supplemented with 2% fetal bovine serum. These cells were evaluated under two experimental conditions: control and submitted to the application of ultraviolet A light and riboflavin 0.1% (CXL) for 30 min. RESULTS: Patients with KC displayed increased levels of Gal-1 and Gal-3 in conjunctival epithelial cells compared with control. Furthermore, KC corneas were associated with intense expression of Gal-1 in the stroma, released by keratocytes. Ultrastructural analysis of keratocytes showed a marked increase of endogenous Gal-3 levels, but not Gal-1, in KC. In vitro, CXL induced significant release of Gal-1 in keratocyte supernatants (116±18 ng/mL, P<0.05) and decreased inflammatory biomarkers as interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-2 and MMP-9. Gal-3 levels were not detected in the keratocyte supernatants. CONCLUSION: Gal-1 and Gal-3 represent new interesting KC biomarkers as revealed by their different expression patterns in KC and control corneal samples. CXL has an immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased expression of anti-inflammatory protein Gal-1.

Anti-inflammatory effect of galectin-1 in a murine model of atopic dermatitis.

Atopic dermatitis (AD) is caused by both dysregulated immune responses and an impaired skin barrier. Although beta-galactoside-binding protein galectin-1 (Gal-1) has immunomodulatory effects in several inflammatory disorders, therapeutic strategies based on its anti-inflammatory properties have not been explored in AD. Thus, we evaluate pharmacological treatment with Gal-1 in the progression of an ovalbumin (OVA)-induced AD-like skin lesions. The skin of OVA-immunized male BALB/c mice was challenged with drops containing OVA on days 11, 14-18 and 21-24. Additionally, in the last week, a subset of animals was treated intraperitoneally with recombinant Gal-1 (rGal-1) or dexamethasone (Dex). Treatment with rGal-1 decreased the clinical signs of dermatitis in BALB/c mice and diminished local eotaxin and IFN-γ levels. The treatment also suppressed the infiltration of eosinophils and mast cells, which was verified by reduced expression of mouse mast cell protease 6 (mMCP6) and eosinophil peroxidase (EPX). These localized effects are associated with extracellular signal-regulated kinase (ERK) activation and downregulation of endogenous Gal-1. The inhibition of disease progression induced by rGal-1 was also correlated with reduced plasma IL-17 levels. Our results demonstrate that rGal-1 is an effective treatment for allergic skin inflammation in AD and may impact the development of novel strategies for skin inflammatory diseases. KEY MESSAGES: Pharmacological treatment with rGal-1 reduces clinical signs of atopic dermatitis. Systemic treatment with rGal-1 inhibits eosinophil and mast cell influx in the skin of AD animals. rGal-1 reduced local eotaxin levels and systemic IL-17 levels. The inhibition of disease progression induced by rGal-1 was correlated with upregulation of phosphorylated ERK.